Bacterial surface polysaccharides play central roles in a wide range of biology, and could serve as targets for novel anti-microbial agents, pathogen sensors, vaccine antigens or other important therapeutics. All of these applications require robust methods to produce these materials that can be easily adapted from one type of polysaccharide to another. One important way to go about doing this is to exploit the natural pathways that are associated with the formation of these materials to build them either enzymatically or engineer a living system to do it. Both of these options require more effective tools for the analysis of bacterial polysaccharide biosynthesis and a better understanding of these natural pathways than is currently available. In this proposal we aim to begin the development of new methods and tools for the production and analysis of complex polysaccharide biosynthesis in vitro and in cells. We will start with the Escherichia coli exopolysaccharide colanic acid (CA), which is thought to promote biofilm formation and help the organism survive in low pH environments. We will first utilize a fluorescent bactoprenyl phosphate mimic to reconstitute the biosynthesis of CA in vitro to elucidate the precise biochemical roles of all proteins involved. This system will be used to produce standards that will then allow us to validate a new cellular probe developed to monitor CA biosynthesis in cultured bacteria, and build tagged cellular polysaccharide precursors in mutant E. coli strains. This CA biosynthesis system will then be replaced in E. coli with an operon from the mammalian symbiont Bacteroides fragilis, which is responsible for the formation of capsular polysaccharide A (CPSA). CPSA is thought to play important roles in the normal development of the mammalian immune system, and could be a key therapeutic for autoinflammatory diseases. This operon replacement strategy in the CA biosynthesis locus will provide a system for the production and excretion of this important biomolecule. The tools generated in this proposal will allow for the optimization of the production of this material. The system developed could then be applied to nearly any polysaccharide of this type, in nature, providing a robust genetically encoded factory for polysaccharide production.